FR2872290A1 - Implementation of plurality of parallel enzymatic reaction, used to diagnose e.g. viral infection, comprises obtaining solid support comprising zone matrix, depositing substrate and implementing plurality of the reaction in reactive volume - Google Patents

Abstract

Process of implementation of a plurality of parallel enzymatic reactions in separated reactive volumes, comprises obtaining a solid support comprising a matrix of hydrophilic zones, where each hydrophilic zone is delimited by a hydrophobic zone; depositing or fixing a substrate in each hydrophilic zone; and implementing a plurality of parallel enzymatic reactions in reactive volume and comprising appropriate reactants and buffers. Process of implementation of a plurality of parallel enzymatic reactions in separated reactive volumes, comprises obtaining a solid support comprising a matrix of hydrophilic zones, where each hydrophilic zone is delimited by a hydrophobic zone (which is separated from other hydrophilic zones, hydrophilic and hydrophobic zones) comprising an aqueous solution deposited at the level of an hydrophilic zone forming a drop confined by the surface tension in the hydrophilic zone; depositing or fixing at least a substrate (which react in an enzymatic reaction) in each hydrophilic zone; and implementing a plurality of parallel enzymatic reactions in reactive volume (constituted by drops of aqueous solution confined in each hydrophilic zone of the support) and comprising appropriate reactants and buffers. Independent claims are also included for: (1) process of detection of parallel enzymatic activity in several samples, preferably several biological samples, comprising implementation of the process, in which the reactive mixture of each drop contains a sample (having an enzymatic activity) to be detected; (2) process of high throughput screening of inhibitor or regulators of an enzymatic activity, comprising the implementation of the process, in which the reactive mixture of each drop contains a reactive (having an enzymatic activity) in the presence of a compound, where the inhibiting or regulatory activity of the compound is researched; (3) process of preparation of a support for the implementation of the process, comprising depositing a mask containing glycerol on an hydrophilic solid support comprising glycerol/sucrose/water mixture in proportions such that the solution obtained is compatible with the piezoelectric exemption, in particular in terms of viscosity; and depositing a layer of hydrophobic silane by co-evaporation with pentane in inert atmosphere, under conditions allowing the formation of a layer of hydrophobic silane in the hydrophobic zone; optionally thermally stabilizing or dehydrating the layer of hydrophobic silane under vacuum; and (4) support comprising a matrix of hydrophilic zones, where each hydrophilic zone is delimited by a hydrophobic zone, which is separated from the other hydrophilic zones, the hydrophilic and hydrophobic zones (which is defined when an aqueous solution deposited on the level of an hydrophilic zone forms a drop confined by the surface tension in the hydrophilic zone), where the support comprises at least a substrate of an enzyme which is fixed to the surface in each of the hydrophilic zones.

Description

METHOD OF HIGHLY PARALLEL ANALYSIS OF REACTIONS

ENZYMATICS AND ITS APPLICATIONS

  The invention relates to a new method for the parallel analysis of a plurality of enzymatic reactions. The invention is directed in particular to the fields of diagnosis and high throughput screening of active substances in an enzymatic reaction, in particular the screening of enzymes, substrates, inhibitors or enzyme regulators. The invention relates in particular to a method comprising: a. obtaining a solid support comprising a matrix of hydrophilic zones, each hydrophilic zone being delimited by a hydrophobic zone separating it from the other hydrophilic zones, b. depositing or fixing in each hydrophilic zone at least one substrate capable of reacting in an enzymatic reaction, c. the implementation of a plurality of enzymatic reactions carried out in parallel in reaction volumes constituted by the drops of aqueous solution confined in each hydrophilic zone of the support and containing the appropriate reagents and buffers.

  The enzymes of the protease family function to cleave certain peptide bonds, and they play a key role in the cell. They are indeed involved in important biological processes such as protein processing and degradation, cell signaling, differentiation, apoptosis ... The production of these enzymes, their abnormal functioning is associated with many pathologies ( inflammatory reactions, cancers, pathogen infections, and Alzheimer's disease), making them both therapeutic targets and diagnostic markers of interest ([1] Leung, D., Abbenante, G .; DP Protease Inhibitors: Current Status and Future Prospects, J Med Chem 43: 305 (2000). & [2] Weissleder, R. Ntziachristos, V. Shedding Light Onto Molecular Target Molecules 9: 123 (2003) . 20

  More generally, the detection of the specific activity of enzymes, and especially proteases, or the screening of active molecules on these enzymes is of obvious interest in the fields of medical diagnosis or pharmaceutical research for the discovery of new active substances.

  In order to determine the specific activity of an enzyme, it is necessary to measure the activity of the enzyme under study under various conditions, and in particular by varying the conditions of enzyme and substrate concentrations, and optionally as a cofactor. Similarly, it is necessary, when searching for active molecules, for example molecules interfering with the activity of enzymes, to observe the effect of a large number of molecules, in particular inhibitors, generally several hundred , at various concentrations. Ideally, all of these conditions should be tested simultaneously and using the lowest possible volumes to achieve a fast result while minimizing the costs associated with such screening methods. A method combining all these characteristics would therefore be particularly useful.

  Today, there are many in vitro macroscopic methods developed for enzymology. In particular, the methods described in the patent application WO 02074997 (QTLBiosystems) and in the patent application WO 0194332 are mentioned.

  Schematically, we can divide into four groups, the current macroscopic methods of detection of proteolytic activity: a) those using a protein substrate analyzed on gel electrophoresis (PAGE), b) those using a peptide substrate analyzed by HPLC chromatography, c ) those using a chromogenic peptide substrate analyzed using a spectrophotometer optionally with a microplate reader, d) those using a fluorogenic peptide substrate analyzed using a fluorescence scanner or a spectrofluorometer with possibly a microplate reader.

  The first two methods are not suitable for the requirements of high throughput screening. Methods using chromogenic or fluorogenic substrates to demonstrate the activity of the enzyme are used with current high throughput screening systems (96 or 384-well plates).

  These systems are not yet satisfactory. In particular, they are expensive, bulky and require handling large amounts of product. The volumes for each microplate enzymatic reaction are at least 100 nL or even much higher.

  Miniaturized devices, grouped under the generic term of protein chips, have recently been developed, more particularly for the analysis of protein interactions with ligands, such as the device described in US Pat. No. 6,630,358 (Zyomyx). These devices are, however, unsuitable for enzymology because of the immobilization of enzymes that disrupts their active conformation.

  Patent application WO2004039487 describes an improved system where proteins are trapped in a sol-gel type matrix.

  The device developed by Biacore allows the analysis of molecular interactions (protein-proteins or protein-ligands) (WO2004 / 038415 Al) from low volumes, however it is not suitable for the parallel analysis of a large number of enzymatic reactions.

  Similarly, enzymatic reaction analysis systems based on the use of microfluidics have been described, in particular in US Pat. No. 6,632,629 (Caliper): The enzymatic reaction is then carried out in a channel where the flow of reagents meet. However, in a single channel, the reactions can only be done in series and the parallelization of the channels is very difficult. This approach therefore makes it impossible to simultaneously monitor several hundred to several thousand enzymatic reactions.

  Highly parallel analysis systems using low volume microwells have been described by Alderman J.C. et al (WO 03059518). A system for contacting a matrix of proteins and chemicals is described by Brennan T.M (US 6,210,894).

  These two approaches have two limitations in common: - the matrix is closed by a hood; the alignment of the covers with the dies can be a difficulty when they are put in place, - once the cover is put in place, it is impossible to add another reagent afterwards.

  There is therefore a real need to develop new microtechnological methods for measuring the activity of an enzyme and the influence of compounds on this activity, and to analyze in parallel a large number of enzymatic reactions using very low reaction volumes, which are economical, automatable and easy to implement.

  Schaack et al. have described a virtual well cell culture system using a solid support having hydrophilic zones delimited by a hydrophobic zone, so that a cell culture medium deposited on a hydrophilic zone of the support forms a drop confined in the hydrophilic zone. Such a support makes it possible to perform in parallel thousands of cell cultures in very small volumes.

  To the knowledge of the Applicant, the use of such a support has never been described or suggested for the implementation of a large number of enzymatic reactions.

  Compared to the methods of the state of the art, the method of the invention has many advantages, and allows in particular: - to work on a reduced surface in several hundreds to several thousand separate drops.

  to carry out enzymatic reactions independent of each other in each of the drops; to measure the enzymatic activity (or its inhibition) in all the drops simultaneously.

  More particularly, it makes it possible to test different conditions in parallel: - different enzymes - different concentrations of enzymes and substrates - different inhibitors, regulators or enzyme substrates (peptides, peptide derivatives, small molecules) different concentrations of inhibitors of regulators or enzyme substrates.

  The method of the invention also makes it possible to carry out dynamic measurements in order to determine the evolution of the enzymatic activity over time. The subject of the invention is therefore a process for carrying out a plurality of enzymatic reactions in parallel in separate reaction volumes, said method comprising: a) obtaining a solid support comprising a matrix of hydrophilic zones, each hydrophilic zone being delimited by a hydrophobic zone separating it from the other hydrophilic zones, the hydrophilic and hydrophobic zones being characterized in that an aqueous solution deposited at a hydrophilic zone forms a drop confined by the surface tension in said zone; hydrophilic, b) depositing or fixing in each hydrophilic zone at least one substrate of an enzymatic reaction, c) carrying out a plurality of enzymatic reactions in parallel in a reaction volume consisting of a drop of aqueous solution confined in each hydrophilic zone of the support and containing the appropriate reagents and buffers.

  In a preferred embodiment of the method according to the invention, it further comprises a step d. comprising detecting in parallel the enzymatic activity in each reaction volume and preferably measuring it.

  The measurement of the enzymatic activity can be carried out by any method known to those skilled in the art; in particular by detection of chromogenic substrates with a spectrophotometer associated with a microplate reader, by immuno-detection, by surface plasmon resonance or SPR (Danielson, Eur J Pharm Sci 2001 May, 13 (2): 203-12) with possibly a slight modification of the support. In particular, step d. is carried out by detecting during the enzymatic reaction and / or at the end of said reaction the amount of a compound whose fluorescence is revealed or extinguished by the enzymatic reaction.

  Such compounds include, in particular, fluorophore substrates of the fluorophore type (J Am Chem Soc 2002 Dec 18, 124 (50): 14868-70; J Org Chem 2002 Feb 8 67 (3): 910-5; PNAS USA 2000 Jlu 5; 97 (14): 7754-9), FRET (Biopolymers 2002, 66: 93-100, Nat Biotechnol 2000 18: 1071-4, Chem Commun 2002 21: 2092-3), SQ (Molecular Probes & Neoplasia). 2002 4: 228-36 and DA (Anal Chem 2004, 76, 1429-1436 (PerkinElmer), PNAS USA 2004, 101: 7511-5, Epub 2004 May 10 (QTL Biosystem)). 1-fluorophores consist of the part P linked to a molecule which has the particularity of becoming fluorescent when it is separated from the molecule by a cleavage enzyme (for example a protease in the case of a peptide bond). principle is similar for DA (donor-acceptor) and FRET (Fluorescence Resonance Energy Transfer) substrates: a molecule is fixed on the P part and it does not emit fluorescence as long as it is in the vicinity of a second one molecule attached to the second part P 'linked to the part P. When the parts P and P' are separated by action of the enzyme, the molecule attached to P becomes fluorescent.

  As regards the substrates SQ (Self-Quenched), the two molecules involved are identical, and a high density of substrate (hooked on a polymer for example) makes it possible to obtain a decrease in the fluorescence. After cleavage, the fluorescence increases again. This type of substrate SQ is also usable in solution in the case of protein substrates labeled with a fluorescent molecule in large quantities.

  Among the substrates that can be used to reveal a protease-type activity, the following substrates will be used: DQ Gelatin (Molecular Probes), the fluorogenic peptide substrates marketed by Bachem or Molecular probes. Other examples of suitable fluorogenic substrates for helicases, phosphatases and proteases are described in Anal. Chem. 2004, 76: 1429-1436. There are also suitable fluorogenic substrates for esterases, various types of oxidase, enzymes involved in the metabolism of sugars (galactosidase, amylase ...); enzymes involved in phosphorus metabolism (kinase, phosphatases ...); enzymes of the cytochrome P450 complex, etc. (marketed for example by Molecular Probes, http://www.probes.com).

  In the presence of the enzyme and of the reaction medium, the transformation of the substrate, detected by emission or quenching of fluorescence, thus makes it possible to demonstrate the activity of the substrate-specific enzymes.

  Naturally, the detection device will be determined according to the mode of revelation of the enzymatic activity chosen by those skilled in the art.

  In one embodiment of the process using fluorogenic compounds, the amount of fluorescence emitted in each reaction volume can easily be detected by means of a scanner, such as those commonly used with methods using DNA chips or the protein chips (for example a GeneTAC LSIV (PerkinElmer) device.) The fluorescence for each hydrophilic zone is then quantified with a suitable software (for example Genepix Pro 4.0 Axon) .The data is then processed by a spreadsheet (Excel example) allowing to obtain values and graphs.

  If one wishes to carry out kinetic measurements, it is possible to withdraw the support at given times and to measure the fluorescence. It is thus possible to quantify the evolution of the fluorescence over time.

  The solid support used in the process of the invention consists of a plate whose surface is substantially flat, which may be made of silicon, glass or polymer, such as polyurethane, nylon, polyester, polyethylene, polypropylene, polyfluorocarbon, polymethylacrylate (PMMA), polycarbonate, polyvinyl chloride (PVC), polydimethylsiloxane (PDMS) or polysulfone.

  According to the invention, the support used contains a matrix of hydrophilic zones delimited by a hydrophobic zone, the latter being able to be composed of several parts. The term matrix means that the hydrophilic zones are arranged on the support at determined positions, for example in a normed network, in particular orthonormed (forming rows and columns). The hydrophilic and hydrophobic zones on the support are characterized in that an aqueous solution deposited at a hydrophilic zone forms a drop confined by the surface tension in said hydrophilic zone. An example of such a support is shown in FIG. 1. The drop formed in the hydrophilic zone fills all or part of said hydrophilic zone and remains confined thereto.

  Preferably, each hydrophilic zone consists of zones of the substantially flat surface of the support with a size ranging from 5 m2 to 10 mm 2, preferably from 100 m2 to 5 mm 2.

  According to a preferred embodiment, the support contains at least 10 hydrophilic zones, for example at least 100 hydrophilic zones, for example between 10 and 10,000, in particular between 300 and 3000 hydrophilic zones, each hydrophilic zone being capable of containing a reaction volume consisting of a drop of aqueous solution with a volume of between 0.1 and 5000 nl, preferably 1 to 500 nl, for example between 10 and 100 nl.

  In particular, several hundred to several thousand drops may be distributed on a glass slide of standard dimensions (close to 75 mm x 25 mm).

  According to another preferred embodiment, the support used in the process of the invention is a solid support made of glass, silicon, plastic or metal, for example gold or silver, whose surface is treated to obtain a matrix of hydrophilic zones. delimited by a hydrophobic zone, the hydrophilic and hydrophobic zones being characterized by their difference in drop angle greater than 40.

  To impart a hydrophobic or hydrophilic character to the flat surface of the support, it is preferably covered with a hydrophobic or hydrophilic material, such as hydrophobic or hydrophilic silanes, or a hydrophobic polymer layer, for example Teflon.

  A matrix of hydrophilic zones on a hydrophobic bottom (FIG. 1a) can be obtained for example by applying a mask on the surface of the support (optionally pretreated, for example with a mixture of sulfuric acid and oxygenated water) followed by a chemical modification. This mask may be a polymer (for example photoresist), a viscous solution (for example containing glycerol). The mask can be dispensed on the surface by means of a spotter or can be created by photolithography (in particular by the methods described in US Pat. Nos. 5,985,551, 5,474,796 and WO 0216023). The protection of the surface with a mask allows the differential modification of the surface: first unprotected areas for example with a hydrophobic silane (perfluorosilanes, alkylsilanes ...) or with a thin layer of hydrophobic polymer (for example liquid teflon) ) and then, after removal of the mask, deprotected areas with another compound (for example a hydrophilic silane such as 2 [methoxypolyethylenoxy] propyltrimethoxysilane, APTES, bis [2-hydroxyethyl] -3-aminopropyltriethoxysilane).

  Thus, according to a preferred embodiment of the method, the support used is characterized in that the matrix of hydrophilic zones is obtained by means of a chemical modification of the surface, in particular by the localized deposition of hydrophobic or hydrophilic silanes, or the localized deposition of a hydrophobic polymer layer such as teflon, in the hydrophobic or hydrophilic zones. In particular, the chemical modification of the surface is carried out after forming a mask obtained by spotting, by photolithography or by altering the hydrophobic layer by laser or plasma.

  Other examples of production of support containing a matrix of hydrophilic zones are in particular described in patent FR 0209326 (Schaack et al.) Since the support makes it possible to confine the microdroplets in the matrix of hydrophilic zones, it is not possible to necessary to fix the substrates on the support, the substrate of each enzymatic reaction is then in solution in the reaction volume.

  However, under certain conditions, it may be advantageous to fix the substrate on the surface of the support or to a gel or a hydrogel, in particular in order to be able to carry out washes before the analysis, and to measure the enzymatic activity. under various conditions. Finally, the simple deposition of substrates in a gel or a hydrogel filling the hydrophilic zones is also possible.

  In a particular embodiment of the process of the invention, the substrate of each enzymatic reaction is attached to the surface of the support. Preferably, the substrate is bonded to the surface, via a spacer, either covalently or by adsorption. The spacer is grafted or adsorbed to the hydrophilic matrix, and allows the compounds to be kept away from the surface while presenting the substrates to the enzymes.

  Examples of usable spacers include artificial polymers such as polyethylene glycol or polyacrylamides or natural polymers such as proteins, sugars or nucleic acids.

  In particular, it is possible to fix on this spacer covalently or by adsorption so-called fluorogenic substrates. In particular, the four types of fluorogenic substrates P'1-Fluorophore, FRET, SQ and D-A which have been defined previously are mentioned. Fixing the substrates to the support advantageously makes it possible to wash the support after the enzymatic reactions have been carried out and to preserve the impression on the plate of the enzymatic modification of the substrates fixed on the plate. The plate thus modified can be analyzed later and the results compared compared to a reference image.

  The deposition of all the reagents in each hydrophilic zone is done by means of appropriate automated system, in particular the Sci-vlexirrayer piezoelectric dispensing robots from Scienion and Nano-plotter from Gesim.

  The substrates deposited in each hydrophilic zone may be identical or different from one hydrophilic zone to another. Where appropriate, at least two different substrates are deposited in the same hydrophilic zone.

  According to a preferred embodiment of the method of the invention, the drops of aqueous solution containing the substrate are deposited at each hydrophilic zone by means of a robot with a piezoelectric ejection system, by contact or soaking. This robot makes it possible to deposit, at specific locations, in the selected hydrophilic zones of the solid support, known quantities of substrates. This same robot can also deposit all the reagents and buffers necessary for carrying out the enzymatic reactions.

  The method of the invention allows in particular the detection of an enzymatic activity in several samples in parallel, in particular several biological samples, in particular at least 10, for example between 10 and 10,000, in particular between 300 and 3000 samples. In this application, the reaction mixture of each drop contains a sample capable of exhibiting the enzymatic activity to be detected.

  The samples are deposited in each hydrophilic zone using a suitable device, for example a piezoelectric injection robot, independently of the substrate, or concomitantly. In order to limit evaporation, glycerol (from 1 to 25% by volume) can be added to the samples containing the reagents. It is also possible to lower the temperature of the deposition surface and the reagents and / or to increase the humidity level in the chamber of the device used for depositing the samples.

  The control of the evaporation of the drops can be ensured (in addition to the presence of glycerol in the microdroplets) by the use of an enclosure making it possible to maintain a high humidity level for several days.

  According to a preferred embodiment, the method allows the parallel detection of an enzymatic activity in a plurality of biological samples capable of having an enzymatic activity detectable by the method of the invention.

  By biological sample, it is necessary to understand all biological material and in particular, a sample of cells, a protein extract of cells, a sample of tissues, organs, biological fluids such as blood, urine, saliva or else the cerebrospinal fluid.

  The biological sample can be prepared from cells of a biological organism, including a sample of tissues or biological fluids such as blood, cerebrospinal fluid, urine or saliva. The sample may be taken from an animal (in particular a mammal and preferably a man) or a plant. The sampling can in particular be performed on a healthy individual or a patient suffering from a pathology. The pathology can in particular be a cancer, a neuro-degenerative pathology, an infectious pathology and in particular a viral, bacterial or parasitic pathology. The biological sample can also be taken from a culture of eukaryotic or prokaryotic cells, bacteria, fungi or yeasts. It can also be taken from an agri-food product, including seeds, fruits, cereals or cooked foods.

  For the purposes of the invention, the term "sample" is also intended to mean any composition capable of producing an enzymatic reaction detectable by the process of the invention in the presence of a suitable substrate. A sample may in particular contain a purified enzyme in an appropriate buffer and, where appropriate, one or more other necessary reagents (co-factors, co-substrate, catalysts, etc.). The sample may contain synthetic or natural chemical compounds.

  The method of the invention allows in particular the detection of any enzymatic activity detectable by means of fluorogenic substrates, by emission or extinction of fluorescence. In particular, it allows the detection of enzymatic activity of DNA repair enzymes, enzymes involved in metabolism and energy cycling (ATPase), or even the detection of enzymatic reactions in cascades (such as the ubiquitin proteolysis system). -dependent), kinase, phosphorylase type, especially those involved in signaling pathways.

  In a preferred embodiment, the method of the invention makes it possible to detect a protease enzyme activity. The proteases detected according to the method are in particular viral, bacterial, metalloprotease or extracellular matrix metalloprotease (MMPs) proteases.

  The method of the invention has applications more particularly in the fields of medical diagnosis.

  In a preferred embodiment, the sample or samples capable of exhibiting enzymatic activity are biological samples taken from an individual, preferably from humans, and the detection of an enzymatic activity in the sample enables the diagnosis to be made. the prognosis and / or the follow-up of the evolution of a pathology or a viral, parasitic or bacterial infection or allows the follow-up of the treatment administered to a patient.

  Within the meaning of the invention, pathology is understood to mean any disorder or dysfunction of the human or animal body. The measurement of the enzymatic activity can be associated with the presence of a pathology, in particular of an infection (diagnosis) or also associated with a level of evolution (prognosis) of a pathology, in particular of a pathology of Viral origin or cancer.

  The method of the invention makes it possible, for example, to determine whether an active substance inhibits the activity of proteases involved in specific pathologies or not. Examples of proteases involved in specific pathologies are in particular given in Table 1 on page 307 of the Journal of Medicinal Chemistry, 2000, Vol. 43, No. 3.

  The method of the invention makes it possible in particular to detect the enzymatic activity of proteases involved in an infection with an HIV-type virus, HBS, for example in a pathology such as hepatitis C or cancer, the enzymatic activity of these proteases being used as a diagnostic marker. In addition, it can be used in the search for enzyme inhibitors produced during the development of these pathologies and which promote their establishment or development. In particular, inhibitors of the hepatitis C virus NS3 protease or to observe the activity of metalloproteases of the extracellular matrix, some of which are related to the development of cancers, can be investigated.

  More generally, the invention relates to the use of the method as defined above, for the diagnosis of a pathology or a viral, bacterial or parasitic infection.

  The method of the invention also has applications more particularly in the field of high throughput screening for the search for active substances. This type of screening mainly concerns the pharmaceutical industry.

  The invention aims in particular at a method of high-throughput screening of inhibitors or regulators of an enzymatic activity, comprising the implementation of a method according to the invention as described above, in which the reaction mixture of each drop contains a reagent capable of exhibiting an enzymatic activity in the presence of a candidate compound whose inhibitory or regulatory activity is desired.

  More generally, the invention relates to the use of the method for carrying out a plurality of enzymatic reactions as defined above, for the high-throughput screening of enzymes, inhibitors or regulators of an enzymatic activity. or enzyme substrates.

  The invention also relates to a method for obtaining a support suitable for carrying out the processes of the invention, said method comprising: a) the deposition on a hydrophilic solid support of a glycerol-based mask, comprising a glycerol / water mixture in such proportions that the solution obtained is compatible with the piezoelectric exemption, especially in terms of viscosity, and b) the deposition of a layer of hydrophobic silanes by co-evaporation with pentane in an inert atmosphere, in conditions allowing the formation of a layer of hydrophobic silanes in the hydrophobic zone; c) optionally, thermal stabilization or vacuum dehydration of the layer of hydrophobic silanes.

  According to a particular embodiment, the hydrophilic solid support is a glass blade cleaned with piranha or silicon oxide surface.

  The invention also relates to a support comprising a matrix of hydrophilic zones, each hydrophilic zone being delimited by a hydrophobic zone separating it from the other hydrophilic zones, the hydrophilic and hydrophobic zones being defined in that an aqueous solution deposited at the level of a hydrophilic zone forms a drop confined by the surface tension in said hydrophilic zone, said support being characterized in that at least one substrate of an enzyme is attached to its surface in each of the hydrophilic zones.

  Preferably, the substrate is bonded to the surface of the support via a spacer as described above. In particular, the substrate is a fluorogenic peptide substrate and allows the fluorescence detection of a protease type activity.

  Such a support can be used in particular in the method of implementing a plurality of enzymatic reactions according to the invention.

  The examples which follow make it possible to illustrate certain preferred embodiments of the method of the invention and to more easily understand its implementation, without however limiting the scope of the invention.

DESCRIPTION OF THE FIGURES

  Figure 1: Figure 1 shows an example of support used in the method of the invention. A. Example of layout of the hydrophilic zones in an orthonormal network. B. Example of chemical modification of the surface of the support with perfluorinated silane C. Example of chemical modification of the surface using a thin layer of Teflon.

  Figure 2: Figure 2 is a photograph of the supports on which are deposited the reaction volume drops with a piezoelectric injection robot.

  Figure 3: Figure 3a shows the arrangement of the drops on the support for the parallel analysis of the activity of a protease.

  FIG. 3b is a graph representing, for each group of drops harboring the same reaction, the fluorescence intensity detected as a function of time.

  Figure 4: Figure 4 is a graph showing the effect of different substrate concentrations on the measured fluorescent signal (10 mg / ml, 100 mg / ml, 250 mg / ml and 500 mg / ml, respectively) as a function of time (40, 60, 80, 120, 160, 200, 240 min) and with or without inhibitor.

  FIG. 5: FIG. 5 is a graph showing the effect of different protease concentrations on the fluorescent signal obtained (respectively 10 mg / ml, 100 mg / ml, 250 mg / ml and 500 mg / ml) as a function of time (40, 60, 80, 120, 160, 200, 240 min) and with or without inhibitor.

EXAMPLES

  EXAMPLE Preparation of a Support Which Is Suitable for the Process of the Invention Comprising a Matrix of Hydrophilic Zones The matrix of hydrophilic zones on a hydrophobic bottom is obtained by the use of a glycerol-based mask, followed by a deposit of hydrophobic silanes in the gaseous phase under an inert atmosphere.

  The method used comprises the following steps: 1. Deposit of a glycerol / sucrose / water mixture mask (for example 15% by weight of glycerol, 25% by weight of sucrose, supplemented with water) on a Hydrophilic solid support (piranha-cleaned glass slide consisting of a 1: 1 mixture of sulfuric acid-oxygenated water, surface-oxidized silicon, etc.) 2. Deposition of a layer of hydrophobic silanes (for example, perfluorooctyltriethoxysilanes) by co-evaporation with pentane in an inert atmosphere (desiccator under argon for example).

  3. Overnight oven at 115 ° C for about 2 hours.

  4. Washing the mask with water This method has the advantage of being fast and easy to implement compared to methods using photoresist-type materials.

  EXAMPLE 2 Example of Implementation of the Method of the Invention for the Detection of a Proteolytic Activity in the Presence of Different Concentrations of Inhibitors In this example, the photolithography method developed by Butler et al (Am. Chem) was used. Soc., 2001, 123: 8887) to create a support consisting of a glass slide comprising a matrix of hydrophilic spots bound to a perfluorinated surface (see FIGS. 1A and 1B). The differences in surface tension are such that an aqueous solution deposited in a hydrophilic zone remains confined in the defined hydrophilic zone. Supports having between 300 and 3000 hydrophilic spots (about 500 μm in diameter) have been used in the present method of detecting protease activity (see FIG. 2) Drops of about 20 nI containing the reagents have been deposited. on the surface of the glass slides. A robot (Sciflexarrayer, Scienion company) was used to deposit the reagents on the glass slides at a defined hydrophilic spot, so that the supports make it possible to test a large number of different conditions in a single reaction.

  The following were carried out as follows: The protease, the fluorogenic substrate, here Self-Quenched gelatin (denoted gelatin SQ) (DQ Gelatin, Molecular Probes), in a buffer containing 10% glycerol and, if appropriate, the inhibitor of protease (here 1,10-phenanthroline) are mixed directly in the drops deposited with the piezoelectric injection robot on the support according to the preset deposition scheme. The reaction buffer has the following composition: 0.05 M Tris-HCl, 0.15 mM NaCl, 5 mM CaCl 2, 0.2 mM NaN 3, pH 7.6.

  In particular, different protease concentrations were tested, as well as the presence of an inhibitor. In total, 40 enzymatic reaction conditions (measured on 400 drops) were tested in parallel. The evolution of the reactions over time is followed by release of the fluorescence after cleavage of the substrate by the protease. The fluorescence is measured using a Genetac LSIV fluorescence scanner. The fluorophore is excited at 488 nm and the emission measured at 512 nm.

  Figure 3a shows the type of image obtained.

  After quantification and data processing using Genepix Pro 4.0 software, the kinetics of proteolysis can be precisely established.

  The kinetics of proteolysis and the effect of the inhibitor could be determined in parallel for each individual drop. After data processing, the mean fluorescence values and standard deviations deduced for each type of reaction were collated on the graph presented in Figure 3b. In particular, the activity of the protease is observed thanks to the variation of fluorescence over time. This activity increases with protease concentration.

  The effects of substrate concentration were analyzed (Figure 4), as well as the effects of protease concentration (Figure 5).

  This example shows that the process of the invention provides a quick and simple method for carrying out parallel analysis of numerous enzymatic reactions under different conditions.

Claims (29)

  A method of carrying out a plurality of enzymatic reactions in parallel in separate reaction volumes, said method comprising: a. obtaining a solid support comprising a matrix of hydrophilic zones, each hydrophilic zone being delimited by a hydrophobic zone separating it from the other hydrophilic zones, the hydrophilic and hydrophobic zones being characterized in that an aqueous solution deposited at the level of a hydrophilic zone forms a drop confined by the surface tension in said hydrophilic zone, b. depositing or fixing in each hydrophilic zone at least one substrate capable of reacting in an enzymatic reaction, c. the implementation of a plurality of enzymatic reactions in parallel in reaction volumes consisting of the drops of aqueous solution confined in each hydrophilic zone of the support and containing the appropriate reagents and buffers.   2. Method according to claim 1, characterized in that it comprises step d. comprising detecting, in parallel, the enzymatic activity of the enzymatic reactions conducted in each reaction volume, and preferably measuring it.   3. Method according to claim 2, characterized in that the measurement of the enzymatic activity is obtained by means of the detection during the enzymatic reaction of the quantity of a compound whose fluorescence is revealed or extinguished by the enzymatic reaction. .   4. Method according to claim 3, characterized in that the amount of fluorescence emitted in each reaction volume is detected using a fluorescence scanner.   5. Method according to one of claims 2 to 4, characterized in that the measurement of the enzymatic activity of the enzymatic reactions is carried out at different times during the enzymatic reaction.   6. Method according to any one of claims 1 to 5, characterized in that the solid support contains a matrix of at least 10 hydrophilic zones, for example from 10 to 10,000, in particular between 300 and 3000 hydrophilic zones, each hydrophilic zone. being capable of containing a reaction volume consisting of a drop of aqueous solution with a volume of between 0.1 and 5000 nl, preferably of 1 to 500 nl, for example between 10 and 100 nl.   7. Method according to one of claims 1 to 6, characterized in that the support is a solid support glass, silicon, plastic or metal, for example gold or silver, whose surface is modified to obtain a matrix hydrophilic zones delimited by a hydrophobic zone, the hydrophilic and hydrophobic zones being characterized by their difference in drop angle greater than 40.   8. Method according to claim 7, characterized in that the matrix of hydrophilic zones is obtained by means of a chemical modification of the surface, in particular by the localized deposition of hydrophobic or hydrophilic silanes, or the localized deposition of a layer of hydrophobic polymer, for example teflon, in the hydrophobic or hydrophilic zones.   9. Method according to claim 8, characterized in that the chemical modification of the surface is performed after formation of a mask obtained by spotting, photolithography or alteration of the hydrophobic layer by laser or plasma.   10. Method according to one of claims 1 to 9, characterized in that the substrate of each enzymatic reaction is in solution in the reaction volume.   11. Method according to one of claims 1 to 9, characterized in that the substrate of each enzymatic reaction is attached to the surface of the support.   12. Method according to one of claims 1 to 9, characterized in that the substrate of each enzymatic reaction is present in a gel or hydrogel filling the hydrophilic zones.   13. The method of claim 10, characterized in that a drop of an aqueous solution containing the substrate is deposited at each hydrophilic zone with a robot with piezoelectric ejection system, by contact or soaking .   14. The method of claim 11, characterized in that the substrate is grafted or adsorbed on the surface of the support by means of a spacer, for example artificial polymers such as polyethylene or polyacrylamide or natural polymers such as proteins , sugars or nucleic acids, said spacer for maintaining said substrate away from the surface.   15. A method for detecting an enzymatic activity in several samples in parallel, in particular several biological samples, comprising the implementation of a method according to one of claims 1 to 14, wherein the reaction mixture of each drop contains a sample likely to have the enzymatic activity to be detected.   The method of claim 15, wherein the enzymatic activity to be detected is a protease activity.   17. The method of claim 15 or 16, wherein the sample or samples likely to have an enzymatic activity are biological samples taken from an individual, preferably from humans, and the detection of an enzymatic activity in the sample. allows the diagnosis or prognosis of a pathology or a viral, bacterial or parasitic infection.   18. A method for high-throughput screening of inhibitor or regulators of an enzymatic activity, comprising the implementation of a method according to one of claims 1 to 14, wherein the reaction mixture of each drop contains a reagent. capable of exhibiting an enzymatic activity in the presence of a candidate compound whose inhibitory or regulatory activity is sought.   19. Use of the method according to one of claims 1 to 14 for the diagnosis of a pathology or a viral, bacterial or parasitic infection.   20. Use of the method according to one of claims 1 to 14 for the high-throughput screening of enzymes, inhibitors or regulators of an enzyme activity or enzyme substrate.   21. A process for preparing a support suitable for carrying out a method according to one of claims 1 to 18, comprising: a. deposition on a hydrophilic solid support of a glycerol-based mask, comprising a glycerol / sucrose / water mixture in proportions such that the solution obtained is compatible with the piezoelectric exemption, especially in terms of viscosity; and B. depositing a layer of hydrophobic silanes by co-evaporation with pentane in an inert atmosphere, under conditions allowing the formation of a layer of hydrophobic silanes in the hydrophobic zone; vs. where appropriate, thermal stabilization or vacuum dehydration of the hydrophobic silane layer.   22. The method of claim 21, characterized in that the hydrophilic solid support is a glass blade cleaned with piranha or silicon oxide surface.   23. Support comprising a matrix of hydrophilic zones, each hydrophilic zone being delimited by a hydrophobic zone separating it from the other hydrophilic zones, the hydrophilic and hydrophobic zones being defined in that an aqueous solution deposited at a hydrophilic zone forms a drop confined by the surface tension in said hydrophilic zone, said support being characterized in that at least one substrate of an enzyme is attached to its surface in each of the hydrophilic zones.   24. Support according to claim 23, characterized in that the substrate is bonded to the surface of the support via a spacer.   25. Support according to claim 23 or 24, characterized in that the substrate is a fluorogenic peptide substrate and allows the fluorescence detection of a protease type activity.   26. Support according to one of claims 23 to 25, suitable for carrying out a method according to claim 14.   27. Use of a support according to claim 26, for the implementation of a method according to one of claims 14 to 18.   28. Use of a support according to claim 26, for the diagnosis of a pathology or a viral, bacterial or parasitic infection.   29. Use of a carrier according to claim 26 for the high-throughput screening of enzymes, inhibitors or regulators of enzyme activity or enzyme substrate.