Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/001—Enzyme electrodes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
  • G01N33/5438—Electrodes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00279—Features relating to reactor vessels
  • B01J2219/00306—Reactor vessels in a multiple arrangement
  • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
  • B01J2219/00315—Microtiter plates
  • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00603—Making arrays on substantially continuous surfaces
  • B01J2219/00653—Making arrays on substantially continuous surfaces the compounds being bound to electrodes embedded in or on the solid supports
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
  • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

• the present invention relates to a micro-system with multiple points of chemical or biological analysis implementing a coupling between the probes and a substrate.
• microelectronics is increasingly called upon to be a link in much more complex systems in which several functions are integrated. These systems or micro-systems range from physical sensor applications to the latest development of so-called "biological" chips.
• a sensitive cell capable of measuring a physical phenomenon is associated with an integrated circuit capable of ensuring the processing of information and its exploitation. This is the case with pneumatic safety cushions for the automotive industry (known as an "air bag” in English terminology).
• an integrated circuit undergoes a finishing allowing it to be used in a biological medium. This is the case, for example, with an integrated glucose meter or blood pressure probes.
• the interface between the medium of conventional microelectronics and that of sensors or biologists is the key element of these micro-systems.
• each cuvette is filled with a different DNA probe and the analyte whose genomic sequence is to be known is brought into contact at the time of analysis with all of the cuvettes.
• analytical chemistry also the demand is strong towards the miniaturization of the cuvettes of chemical reactions t
• a first technique consists in activating sites where the reagents will then be deposited and fixed by various chemical molecules. It is a technique mainly used on glass substrates.
• the reagents are deposited by micro-pipetting or by a technique of the inkjet type.
• On the chemical side to ensure the interface between the substrate and the reagent, mention may be made of silanes, lysines, thioles when the substrate is previously coated with gold. This chemistry is complex, especially when it comes to controlling its reproducibility on a substrate that may have a few thousand different sites. Mention may be made, as representative of this technique, of US Pat. No. 5,474,796 which relates to surface structuring: the reagents are fixed to a substrate having hydrophilic zones and hydrophobic zones. The matrixing obtained is therefore very regular.
• the reagent is a DNA probe, in particular an oligonucleotide of around twenty bases
• the probe base after base on each site. It is known to use successive masks to make this synthesis in situ: each site is covered with a photoprotected base. The photomasking then makes it possible to remove the protection from the sites and to chemically attach an additional photoprotected base. The operation is repeated until the desired probe is obtained at each site. It is currently possible to build tens of thousands of different probes on a substrate. This technique is excellent but it does not make it possible to obtain probes with a large number of bases (the limit is approximately 20).
• a third technique relates to electrodeposition on site electrically polarized of a conductive polymer carrying the selected reactive species.
• the substrate is electrically connected to the outside and soaked in a tank containing the chemical species to be deposited.
• the site chosen is polarized and the copolymerization is carried out (in less than a minute at a voltage below I V).
• different reagents were fixed on different areas of the substrate, thus allowing a multipoint analysis.
• each site is provided with a compound organic offering a biotin function.
• Each site can then receive a chemical or biological probe constituted by a determined reagent (a small piece of a DNA strand for example) associated for example with an avidin or streptavidin function.
• a chemical or biological probe constituted by a determined reagent (a small piece of a DNA strand for example) associated for example with an avidin or streptavidin function.
• the coupling which takes place between the avidin and biotin functions ensures the fixation of the probes on their respective sites.
• the subject of the invention is therefore a process for producing a micro-system with multiple points of chemical or biological analysis, comprising the steps consisting in:
• each micro-cuvette being intended to receive a reagent and being provided with fixing means making it possible to attach said reagent to it
• the step consisting in fixing the first organic compound on the means for fixing the micro-cuvettes is carried out by electro-polymerization, the fixing means being electrically accessible conductive means, the hooking function of the first organic compound with the fixing means consisting of a conductive monomer, a counter-electrode being used to carry out the electro-polymerization.
• the conductive means can comprise an electrode common to all the micro-cuvettes.
• the structure can be designed from a substrate, one face of which has said common electrode, a layer of insulating material being deposited on said common electrode, the layer of insulating material being hollowed out up to said common electrode to form the micro-cuvettes whose bottom is constituted by the common electrode.
• the structure can also be designed from an active substrate having a plurality of electrodes on one of its faces, this plurality of electrodes constituting the fixing means, a layer of insulating material being deposited on said face, the layer of insulating material being hollowed out to the electrodes to form the micro-cuvettes, multiplexing means being provided for electrically connecting the plurality of electrodes in order to carry out the electro-polymerization.
• the counter-electrode may be an electrode integral with the structure or be placed opposite the fixing means during the electro-polymerization operation.
• the conductive monomer of the first organic compound is pyrrole.
• the step consisting in fixing the first organic compound on the means for fixing the micro-cuvettes consists in putting using a covalent bond between the attachment function of the first organic compound with the fixing means and the fixing means.
• This covalent bond is for example of silanization type (silane-biotin) or a self-assembly using a thiol-biotin bond.
• the step consisting in fixing the first organic compound to the means for fixing the micro-cuvettes is carried out chemically, by adding a reagent capable of causing polymerization on the means for fixing the first compound organic.
• This first organic compound can be pyrrole and the reagent capable of causing the polymerization of a ferric salt.
• the hooking function of the first organic compound with the second organic compound is a biotin function, the hooking function of the second organic compound with the first organic compound being an avidin or streptavidin function.
• the first organic compound can be a biotinilated pyrrole.
• the second organic compound can consist of said reagent directly attached to the avidin or streptavidin function.
• the step consisting in placing, in each microcuvette, the second organic compound can comprise the deposition of a solution containing the second organic compound in each microcuvette then the elimination of this solution so as to keep only the probes obtained by selective attachment of the first and second organic compounds, for example by binding between biotin and streptavidin / avidin.
• the second organic compound can also consist of said reagent attached to the avidin or streptavidin function via a biotin function.
• the step consisting in placing, in each microcuvette, the second organic compound can comprise the deposition of a first solution of avidin or of streptavidin in each microcuvette, then the elimination of this first solution to reveal the first organic compound with the biotin function complexed by said avidin or said streptavidin, then the deposition of a second solution containing said reagent in biotinilated form, then the elimination of this second solution to keep only the probes obtained.
• the invention also relates to a micro-system with multiple chemical or biological analysis points constituted by a structure provided with micro-cuvettes, each micro-cuvette being intended to receive a reagent and being provided with fixing means allowing attaching said reagent thereto via a first organic compound, the reagent being part of a second organic compound, the first organic compound comprising a bonding function with the second organic compound and a bonding function with the means fixing, these two attachment functions being separated by a spacer, the second organic compound comprising an attachment function with the corresponding attachment function of the first organic compound.
• the fixing means are electrodes and the attachment function with the fixing means of the first organic compound is a conductive polymer.
• the fixing means can form a common electrode.
• the micro-cuvettes can be formed in an insulating layer of said structure, the fixing means forming the bottom of the micro-cuvettes.
• the structure may include a counter-electrode arranged in opposition with respect to the bottom of the micro-cuvettes in order to be able to establish an electric field between the counter-electrode and the fixing means.
• said conductive polymer is a polypyrrole.
• the fixing means and the attachment function of the first organic compound with the fixing means are such that they form a covalent bond.
• This covalent bond is for example of the silanization (silane-biotin) or self-assembly type using a thiol-biotin bond.
• the attachment function of the first organic compound with the fixing means comprises a polymerization reagent, by chemical means, on the fixing means.
• This reagent can be a ferric salt.
• the first organic compound comprises a biotin function for its attachment with an avidin or streptavidin function of the second organic compound.
• the first organic compound can be a biotinilated polypyrrole.
• the second organic compound can consist of said reagent directly attached to the avidin or streptavidin function.
• the second organic compound can also consist of said reagent attached to the avidin or steptavidin function via a biotin function.
• FIG. 1A to 1E show different stages in the production of a structure for a micro-system with multiple points of chemical or biological analysis according to the present invention
• FIG. 2 illustrates a step of electropolymerization carried out in the micro-cuvettes of a structure intended for a micro-system with multiple points of chemical or biological analysis according to the present invention
• FIG. 3 shows a micro-bowl of the structure shown in Figure 2 and at the bottom of which is fixed a first organic compound, according to the method of producing a micro-system according to one invention, -
• FIG. 4 represents another structure intended for a micro-system with multiple chemical or biological analysis points according to the present invention
• - Figure 5 illustrates a step of electropolymerization carried out in the micro-cuvettes of the structure shown in Figure 4;
• - Figures 6 to 8 illustrate the step of placing in a microcuvette the second organic compound to obtain a chemical or biological analysis probe according to the present invention;
• - Figure 9 illustrates the method of manufacturing a first organic compound usable for the present invention.
• Figures 1 to 8 illustrate the alternative embodiment for which the means for fixing the first organic compound are electrodes and for which the placement of this first organic compound is by electro-polymerization, these electrodes allowing the application of a potential electric.
• the structure may include a passive substrate, that is to say that it does not include integrated electronics.
• the substrate may be coated with a conductive plane (for example metallic) itself covered with a layer of a material ensuring the function of electrical insulation and in which the micro-cavities are formed. These lead locally to the driver plane. The exposed areas of the conductive plane then constitute the reception electrodes.
• the substrate can also be active, in which case the electronics integrated into it can serve various functions: localized heating of sites, local pH measurement, reading of a fluorescence signal, etc. In most cases, it is not possible to short-circuit the sites for the subsequent functions which must remain addressable on each site independently of the others. The multiplexing necessary for these functions can then be used during the process of producing the micro-system. It is indeed possible to address all the sites collectively to carry out the operation of fixing the reagents. Each site may subsequently be addressed individually.
• Figures 1A to 1E are cross-sectional and partial views. They illustrate a first embodiment of a micro-system according to the invention for which the counter-electrode is situated on the surface and for which the substrate is passive.
• FIG. 1A represents a substrate 1 constituted by a wafer which can be made of a material such as glass, silicon, plastic. On a main face of this wafer, a metallic layer 3, for example in chromium, gold or platinum, with a thickness of between 0.1 and 10 ⁇ m, has been deposited. As shown in FIG. 1B, a photosensitive polymer film 5, for example a polyimide film with a thickness of between 1 and 50 ⁇ m, is deposited on the metal layer 3.
• a photosensitive polymer film 5 for example a polyimide film with a thickness of between 1 and 50 ⁇ m
• Micro-cuvettes 7 are then formed by exposure and development of the polyimide film (see FIG. 1C). They are advantageously formed with sloping sides. The micro-cuvettes formed locally reveal the metal layer 3. A new metal layer 9 is then uniformly deposited on the polyimide film including the interior of the micro-cuvettes 7.
• the metal layer 9 can be made of chromium, gold or platinum and be 0.1 to 10 ⁇ m thick.
• a layer of masking resin 11 is deposited on the metal layer 9 and areas to be etched in this metal layer 9 are defined.
• Each micro-bowl 7 has at its bottom an electrode 9a, all the electrodes 9a being electrically connected by means of the metal layer 3.
• a common electrode 9b covers the upper face of the polymer film 5. This structure is ready for the next step consisting in placing the first organic compound at the bottom of the micro-cuvettes.
• FIG. 2 illustrates the electropolymerization step.
• a structure 20 provided with micro-cuvettes 21 and which, unlike the case described above, is formed from an active substrate 22.
• the micro-cuvettes 21 have been formed in an insulating layer 23 deposited on the substrate 22
• the bottom of the micro-cuvettes 21 is constituted by the electrodes 24 which can be, by multiplexing, electrically connected.
• a common counter electrode 25 has been deposited on the upper part of the insulating layer 23. A potential difference can therefore be applied between the counter electrode 25 and the electrodes 24 electrically connected by multiplexing.
• a layer 26 containing suitable monomers for example a mixture of pyrrole and biotinylated pyrrole like that described below, is deposited on the face of the substrate 20 having the micro-cuvettes.
• An appropriate potential difference is applied between the counter electrode 25 and the electrodes 24.
• the micro-cuvettes are rinsed and dried.
• An organic compound 30 is then obtained, as shown in FIG. 3, consisting of biotinilated polypyrrole, fixed on the electrodes 24. This organic compound adheres to the electrodes 24 by the pyrrole cycles and presents biotin molecules 31 attached to pyrrole cycles by spacers 32.
• the structure intended to constitute the microsystem according to the invention may not include a counter electrode directly deposited on itself.
• the structure 40 comprises for example a passive substrate 41, one face of which is covered with a metal layer 42 forming an electrode.
• An insulating layer 43 has been deposited on the metal layer 42 and etched to form micro-cuvettes 44.
• the electro-polymerization is then carried out, as shown in FIG. 5, in a tank 45 filled with an appropriate solution 46 containing a mixture of pyrrole and biotinilated pyrrole monomers.
• a DC voltage generator 47 is connected between the electrode 42 and a counter-electrode 48 arranged opposite the bottom of the micro-cuvettes 44.
• the electropolymerization causes the deposition of biotinilated polypyrrole on the visible areas of the electrode 42 as for FIG. 3.
• FIGS. 6, 7 and 8 illustrate the fixing of the second organic compound.
• the electropolymerization tank 45 may include an additional electrode serving as a reference.
• the next step in the process is represented in FIG. 6. It consists in placing, in each microcuvette, a solution 50 comprising the second organic compound 51.
• This second organic compound comprises for example an avidin or streptavidin function if the first compound organic is biotinilated.
• a DNA probe can thus be constituted by choosing as the second organic compound DNA streptavidin which is marketed.
• Biotin / streptavidin recognition is widely used industrially because of the high recognition rate, insensitivity to temperature variations, a large choice of buffer, pH, etc.
• FIG. 7 shows the attachment carried out between the biotin function of the organic compound 30 and the avidin or streptavidin function of the organic compound 51. After rinsing and drying, a micro-system is obtained, an analysis point of which is shown in FIG. 8.
• the micro-cuvette 21 is provided with probes formed by the association of organic compounds 30 and 51.
• DNA, RNA, oligonucleotides, enzymes, nucleic or amino acids, proteins, antigens or any product which can be linked to 1 ′ can be attached to the biotinilated polypyrrole at the bottom of the microcuvettes.
• avidin or streptavidin
• biotin can form not only the association: polypyrrole-biotin / (strept) avidin-reactive, but also the association: polypyrrole-biotin / (strept) avidin / biotin-reactive.
• Biotinilated DNA which is a commercial product.
• Biotin / streptavidin / biotin double recognition has the same qualities as the biotin / streptavidin recognition reaction.
• the invention allows collective placement of the first organic compound on the bottom of the microwells.
• the biotinilated polypyrrole which clings to the bottom of the microcuvettes is advantageously a copolymer synthesized from pyrrole and biotinilated pyrrole for which the biotin is attached to the nitrogen atom of a pyrrole cycle by a hydrophilic spacer.
• This spacer can be crucial. It must be long enough to ensure unrestricted recognition of biotin by avidin. A length of at least eleven atoms is particularly suitable.
• Figure 9 is a diagram showing the synthesis of a biotinilated pyrrole. This summary is obtained by coupling an aminoalkylpyrrole and a biotin entity. The synthesis of the aminoalkylpyrrole was carried out using a reaction as described by JIRKOWSKY and BAUDY in Synthesis, 1981, page 481. The reagents used and the conditions of the reactions are as follows: i) 4,7,10-trioxa- 1.13-tridecanediamine (3 eq.), acetic acid / dioxane at reflux, for 5 h, yield 32%; ii) 10% KOH at reflux for 5 h, yield

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• 1998
  • 1998-10-13 FR FR9812800A patent/FR2784466B1/fr not_active Expired - Fee Related
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