Classifications

• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/28—Electrolytic cell components
  • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
  • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
  • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
  • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
• • H—ELECTRICITY
  • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
  • H05K—PRINTED CIRCUITS; CASINGS OR CONSTRUCTIONAL DETAILS OF ELECTRIC APPARATUS; MANUFACTURE OF ASSEMBLAGES OF ELECTRICAL COMPONENTS
  • H05K3/00—Apparatus or processes for manufacturing printed circuits
  • H05K3/40—Forming printed elements for providing electric connections to or between printed circuits
  • H05K3/4007—Surface contacts, e.g. bumps

Definitions

• the present invention relates to a microsystem for biological analyzes, usable in particular in the health sector, the food industry and the environment.
• microtechnologies lead to new solutions, both technical (miniaturization, integration) and economic (mass production), likely to boost the development of biosensors.
• FR-A-2 598 227 and 4: EP-A-244 326 also describe a method for detecting and / or identifying a biological substance in a liquid sample using electrical measurements.
• the sample is brought into contact with a reagent-carrying plate comprising a ligand specific for the biological substance to be detected, this plate possibly being made of a semiconductor material such as silicon, and being coated with a silica insulating layer, then we measure the C and / or R components of the electrical impedance of the system to detect the presence of the biological substance in the sample.
• the present invention specifically relates to a biological analysis microsystem, based on the same principle, that is to say on the recognition reaction of the biological substance or analyte to be detected with a specific ligand, which makes it possible to perform the measurement with a distance between the measurement electrodes much more. low and thereby improve the sensitivity of the device.
• the device for detecting an analyte comprises: an insulating support coated with a first conductor forming a first electrode, and a second conductor supporting a plurality of conductive elements extending above the first conductor so as to form a second coplanar electrode arranged at a distance d from the first driver, and
• At least one of the electrodes are covered with a specific ligand L of the analyte to be detected.
• the analyte A possibly present in this sample will react with the ligand L to form an LA complex, ie an active layer. on the electrodes.
• the formation of this layer can be detected by measuring the impedance between the electrodes.
• the particular coplanar structure of the electrodes makes it possible to arrange the two electrodes at a very short distance d, for example from 20 to 500 nm, which is of the order of 2 to 5 times the thickness of the active layer formed by reaction of the analyte with the specific ligand on the electrodes. So, . the impedance measurement will relate to the layer itself and not to a layer of air or of fluid not representative of the phenomenon of recognition between the analyte and the specific ligand.
• This particular arrangement of the electrodes on the same support makes it possible to obtain a multiplicity of overlap zones between the first and second electrodes resulting in a large total surface of distributed covering.
• the distribution of the covering surface and the absence of a second support allows the analyte quick access to the electrodes compared to a device where the electrodes are each on a separate support and where the fluid must enter a space between two thick structures. The smaller this space, the more the fluid is hampered, hence problems of access time and homogeneity.
• this arrangement makes it possible to increase the developed surface of the electrodes and thus to obtain, when measuring the impedance of the active layer formed, a high signal / noise ratio.
• the conductive elements produced for example in the form of mushrooms, have good mechanical strength, which makes it possible to preserve the distance between the electrodes.
• the first conductor has the shape of a comb with wide teeth
• the second conductor has the shape of a comb with narrower teeth, the teeth of the second conductor being interposed between the teeth of the first conductor and supporting conductive elements extending above the teeth of the first conductor.
• the device of the invention can be used for the detection of analytes of various types.
• analytes in particular in the medical field, mention may be made of antigens, haptens, antibodies, peptides, nucleic acid fragments (DNA or RNA), enzymes and enzyme substrates.
• at least one of the electrodes of the device is covered with a ligand specific for the analyte to be detected, for example by direct or indirect grafting of this ligand on the electrode (s) .
• an analyte-ligand complex or active layer, is formed on the electrode (s), and the presence of this complex is directly detected by an electrical impedance measurement.
• the specific ligands covering the electrode (s) are those which have at least one site for recognition of the analyte and which are capable of binding to the latter.
• the ligand-analyte pair can thus belong to the antigen-antibody, hapten-antibody, hormone receptor, DNA- C DNA, RNA- C RNA, enzyme-substrate, or any other association of biological molecules or not, capable of forming between they are complex.
• the invention also relates to a method of manufacturing the device described above.
• This process comprises the following steps: a) forming on an insulating support a first conductor and a second conductor spaced apart from each other, b) coating the insulating support comprising the first and second conductors with an insulating layer, c) etch this insulating layer to expose areas of the second conductor which will serve as electrical contact pads, d) forming on these zones and above the insulating layer conductive elements by galvanic growth of a metal, and e) removing the insulating layer by dissolving in a solvent, dissolving neither the first and second conductors, nor the metal conductive elements.
• steps d) and e) of the process defined above are replaced by the following steps: d ') depositing on the assembly a metallic layer, then a photolithography resin layer, e') exposing the resin and develop it to expose areas of the metal layer having the dimensions of the conductive elements to be formed, f ') forming on these areas of the metal layer the conductive elements by galvanic growth of a metal, g') removing the resin and the metal layer except on the places which correspond to the conductive elements, and h ') remove the insulating layer by dissolving in a solvent, neither dissolving nor first and second conductors, nor the metal of the elements.
• an insulating support in glass or an insulating support in silicon.
• the first and second conductors are preferably formed by depositing metal on the insulating support at the corresponding places. This can be done by metallization followed by etching to define the conductive areas corresponding to the first and second conductors.
• the first second conductors are preferably formed on this support by implantation of ions, for example boron or phosphorus ions at the desired locations.
• an insulating layer for example of silica, is then deposited on the support comprising the first and second conductors, which is then etched to define the contact pads of the conducting elements which will form the second on the second conductor. electrode.
• a metal such as gold.
• a complementary step of coating the first conductor and the conductive elements, ie the electrodes of the device is generally carried out with a specific ligand for the analyte to be detected.
• the embodiment of this step depends, on the one hand, on the material constituting the electrodes and, on the other hand, on the specific ligand used.
• Figure 1 is a perspective view of a detection device according to the invention.
• FIG. 2 is a view in vertical section of the device of the invention along the line XX ′ in FIG. 1.
• Figures 3 to 7 illustrate the main stages of manufacturing the device of Figure 1 in the case where the insulating support is a glass.
• FIGS. 8 to 12 illustrate an alternative embodiment of the manufacturing process illustrated by FIGS. 3 to 7.
• Figures 13 to 17 illustrate another embodiment of the device of Figure 1 suitable for the use of a silicon support.
• Figure 1 there is shown in perspective a microsystem for biological analysis according to one invention.
• This microsystem comprises an insulating support 1 provided on its upper surface with a first conductor 3 and. of a second conductor 5.
• the first conductor 3 which constitutes one of the electrodes of the microsystem, has the shape of a comb whose teeth 3a are relatively large compared to the space between successive teeth.
• the second conductor 5 also has the shape of a comb comprising teeth 5a interposed between the teeth 3a of the first conductor 3, these teeth 5a are narrower than the teeth 3a and they support conductive elements 7 having the form of fungi which s 'extend above the first conductor 3 at a distance d therefrom.
• the conductive elements 7 form the second electrode of the microsystem.
• the electrodes 3 and 7 can be connected to an external electrical circuit respectively by the conductors 13 and 15 in order to polarize the electrodes and to perform impedance measurements between them.
• Figure 2 which is a vertical section of the device of Figure 1 along line XX ', we see that the conductive element 7 in the form of a mushroom is disposed at a distance d from the teeth 3a of the first conductor 3, being supported by the teeth 5a of the second conductor.
• the shape of the fungus makes it possible to have a large electrode surface for the recognition reaction of the analyte to be detected.
• FIGs 3 to 7 there is shown schematically the embodiment of the microsystem of Figure 1 starting from an insulating glass support.
• the production is carried out by a "cold” process, the manufacturing steps being carried out at temperatures not exceeding 300K.
• FIG. 3 illustrates the realization of the first step of the manufacturing process according to which the first and second conductors in the form of combs are deposited on the support 1. This can be done by metallization of the support 1, followed by etching which makes it possible to define the conductive areas which will constitute the first comb-shaped conductor 3 with its teeth 3a and the second comb-shaped conductor 5 with the teeth 5a.
• These combs can be made of gold, a gold-chrome alloy or a gold-nickel-chrome alloy.
• the use of gold or of a gold alloy makes it possible, on the one hand, to then obtain good galvanic growth of the fungi and, on the other hand, to graft under good conditions the electrodes of biological molecules which will constitute the specific ligand of the analyte to be detected.
• step b) an insulating layer 6, for example of silica, is deposited on the coated support obtained in the first step, which is then etched to expose the areas 7a corresponding to the contact pads between the second conductor and the mushroom shaped elements.
• This can be done by cold depositing layer 6, for example by PECVD (chemical vapor deposition activated by plasma) or by spinning with colloidal silica.
• PECVD chemical vapor deposition activated by plasma
• the holes corresponding to the contact pads are then made in layer 6 by photolithography.
• step d) of the method of the invention there is illustrated step d) of the method of the invention.
• the conductive elements 7 are formed in the form of mushrooms by galvanic growth of metal, using the contact pads as deposition electrodes.
• the metal is gold or a gold alloy, it can be carried out at room temperature with a bias voltage of the order of a volt. This galvanic growth directly develops the form of fungus.
• step e) of the method of the invention in which the silica layer 6 is removed by dissolution using hydrofluoric acid.
• the device shown in FIG. 1 is thus obtained.
• this device is subjected to a final step of coating the two electrodes with a specific ligand for the analyte to be detected.
• the surface of the electrodes is modified, for example with thioalkanes when the electrodes are made of gold.
• Figures 8 to 12 there is shown an alternative embodiment of the method described above.
• FIGS. 3 and 4 the first steps of the process are carried out as illustrated in FIGS. 3 and 4, but after etching the layer of silica and obtaining the configuration shown in FIG. 4, a continuous layer of metal is deposited on the support.
• FIG. 8 illustrates this step which can be carried out by sputtering of a metal. In this figure, we see the metal layer 8.
• the assembly is covered with a photolithography resin which will then be hardened by irradiation so as to become insoluble in certain areas of the substrate, the uncured resin being eliminated by an appropriate solvent.
• Figures 9 and 10 show these steps.
• this resin By exposure to the desired areas identified by arrows, this resin is hardened to make it insoluble.
• FIG. 11 illustrates the production of the conductive elements 7. This can be carried out by galvanic growth, for example of gold, under the same conditions as those described above. The structure shown in FIG. 11 is obtained where the elements 7 are delimited in the hardened resin layer 9.
• the step of eliminating the hardened resin layer 9 and the metal layer 8 is shown on the zones which do not correspond not to the conducting elements 7. This can be done by applying an oxygen plasma.
• FIG. 12 The structure shown in FIG. 12 is obtained, which corresponds substantially to that of FIG. 5.
• the step of eliminating the silica layer 6 and the step of grafting a ligand are then carried out as in the case of FIGS. 6 and 7.
• FIGS. 13 to 17 another embodiment of the device in FIG. 1 has been described, using an insulating silicon support.
• the process used is a process of silicon microelectronics type. The manufacturing steps are compatible with the CMOS circuit manufacturing lines.
• FIG. 13 illustrates the first step of the method according to which the first and the second conductors are formed on the support.
• the conductive zones corresponding to the combs 3 and 5 are produced by implanting ions in the silicon support to make it conductive on these zones represented by 3a and 5a in the figure.
• the ions used for implantation are, for example, boron or phosphorus ions, and implantation is carried out through a mask defining the zones to be implanted using sufficient energy to make the silicon conductive on these - zones, over a thickness from a few 1000 ⁇ to 1 micron.
• FIG. 14 illustrates step b) of the method, in which an insulating layer 6 is formed on the assembly.
• This insulating layer is silica which can be formed by thermal oxidation of the silicon on the entire support (implanted zones and not established). After this step, the thickness of the zones implanted is weaker.
• FIG. 9 also illustrates step c) of the method of the invention in which the insulating layer 6 is etched to expose certain areas 7a of the second conductor which will serve as contact pads. This engraving can be carried out by photolithography in. the same conditions as before.
• Figure 15 there is shown the intermediate step of metallization of the contact pads 10 which can be performed by a complete metallization followed by localized etching corresponding to the location of the contact pads.
• FIG. 16 represents the step of forming the conductive elements above the layer 6. This can be carried out by galvanic growth as in the case of FIG. 5.
• FIG. 17 represents step e) of elimination of the silica layer, for example by means of hydrofluoric acid.
• the last step of the process can be carried out as previously by grafting on the conductive elements 7 and on the electrode formed by the first conductor 3 a ligand specific for the analyte to be detected.
• the conductor 3 and the elements 7 are made of gold or of a gold alloy, the surface of the electrodes is modified with thioalkanes, then the ligand is fixed on the gold surfaces thus modified by reaction between the ligand and the terminal chains. of thioalkanes.
• this fixing can be carried out by conventional techniques, for example by adsorption on the electrode after having oxidized it very superficially, or by forming a covalent bond between the electrode and the ligand using a bifunctional coupling reagent capable of reacting both with the electrode and with the ligand.
• reagents suitable for coupling ligands constituted by antibodies, proteins and peptides, on silicon mention may be made of silane derivatives comprising an alkoxysilane group and an NH 2 group separated from one 'other by a hydrocarbon chain. Techniques of this type are described in references 1, 3 and 4 cited above.
• the silicon can be chemically modified by an alkoxy or a chlorosilane comprising a function capable of reacting with the specific ligand, for example a terminal group-CN, -NH2 or -SH.
• these groups are capable of reacting, after activation by an appropriate functional agent, with the groups of molecules such as antibody fragments and oligonucleotides.
• R 1 represents CH 3 , C 2 H 5 , OCH 3 or OCH 2 H 5
• R 2 represents -CN, -NH 2 or -SH
• n is an integer from 1 to 17.
• an oligonucleotide fragment can be attached to this group by conventional reactions.
• R represents SH
• an antibody fragment can also be attached to this group by conventional coupling reactions.
• the device of the invention whose conductive elements 7 and possibly the first conductor 3 have been coated with an appropriate specific ligand, can be used for the detection of analyte in the following manner.
• a drop of the sample to be analyzed is placed on the support plate 1. Given the dimensions of the wafer, the drop covers the elements 7 and the electrode 3. If the sample contains the analyte A, an active layer is formed on the surface of the electrodes 3 a and 7 by formation of the LA complex. which occupies practically the entire thickness d between the electrodes. The presence of this layer is detected by measuring the impedance between these electrodes.

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Citations (2)

• 1996
  • 1996-12-30 FR FR9616201A patent/FR2757949B1/fr not_active Expired - Fee Related
• 1997
  • 1997-12-29 JP JP52970698A patent/JP2001510564A/ja active Pending
  • 1997-12-29 EP EP97953963A patent/EP0948744A1/fr not_active Withdrawn
  • 1997-12-29 WO PCT/FR1997/002440 patent/WO1998029740A1/fr not_active Application Discontinuation

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